Innate immunity provides a first line of defense against pathogen- or danger-associated molecular patterns. Among known innate receptors, Toll-like receptors (TLRs) and NOD-like receptors (NLRs) share a common structural motif that consists of multiple leucine-rich repeats (LRRs) with some variability in amino- acid residues and length of each repeat. Structural studies of LRRs of variable lymphocyte receptors (VLRs) in jawless fish have also shown that LRR domains are important in ligand recognition. Thus, the LRRs of TLRs and NLRs are essential for interaction with agonist ligands in order to initiate cellular signaling in innate immunity. Furthermore, when the LRR domain is disengaged from the signaling domains, it can negatively regulate innate immunity, by competitively binding to ligands. Thus, the ultimate goal of this study is to advance understanding of the structure and function of LRRs in TLRs and NLRs in ligand-induced activation and signaling. Structural and biophysical characterization of LRR domains in TLR1/2/3/4/5/6 have been accomplished by several groups, including our own, but major unresolved questions remain concerning how TLR7/8/9 interact with and are regulated by nucleic acid and other ligands in response to microbial pathogens. Compared to TLRs, structural investigation of NLRs has been extremely limited. To date, only one NLR LRR structure, that of NLRX1, has been solved and we have also shown that it directly binds RNA ligands. With our extensive expertise in expression, purification, crystallization and characterization of diverse LRR proteins, we will investigate structural, biophysical and functional properties of LRR domains in TLR7/8/9 and NLRC3/4/5, as well as advance our already productive studies of TLR5 and NLRX1. Such findings will address the structural and functional mechanisms that arise from the specific interactions of TLR7/8/9 and NLRC3/4/5 with their cognate ligands and lead to understanding of receptor activation and regulation in innate immunity. We will capitalize on our previous studies of TLR5 and NLRX1 to answer key questions about their signaling mechanisms and how they might be regulated by outside agents. Collaborations on cellular aspects on these receptors with Drs. Andrei Gudkov, Cynthia Leifer and Jenny Ting and on specialized biophysical approaches with Drs. Andrew Ward and David Baker will complement our biophysical and functional studies as well as stimulate development of agonists and antagonists as therapeutics in compromised immunity or in hyper- inflammatory diseases.